U.S. Pat. No. 3,888,977, issued on June 10, 1975, to Murray J. Sanders, discloses a method and composition for the treatment of degenerative neurological diseases of the nervous system which, among others, may involve the function of motor nerve cells from their origin to the neuromuscular junction, as well as elements of the central nervous system including axones, nerve myelin sheaths, etc. The present invention provides an improved method of performing the potency test and establishing atoxicity in the compositions of that patent. Hence, the entire disclosure of that patent is incorporated herein by reference and relied upon for the details of producing those compositions and for the method of treatment in connection therewith.
The Sanders patent notes that degenerative neurological diseases progress in a chronic manner to severe physical disability, such as paralysis, and even to death. While the cause of such neurological diseases is not always known, it is believed that the diseases are often caused by virus or proteins with potentially deleterious functions. It is further believed that these noxious moieties cause their degenerating functions by attaching to or involving nerve cell receptors. It is not clear whether nerve cell receptors are discrete anatomical structures of the nerve cell, or are theoretical biophysical concepts which describe on of the functions of the nerve cell. Irrespective, however, nerve cells act as if physical receptors exist in the nerve cell.
As also noted in the Sanders patent, certain neurotropic snake venoms have been shown to involve essentially all of the motor nerve cells, presumably at least in part through the nerve cell receptors, and the basic invention of the Sanders patent is that of blocking the nerve cell receptors by certain detoxified snake venom and thereby interfering with presumed invading pathogenic virus or protein moieties with potentially deleterious functions (or other moieties which may be indeed the cause of the neurological disease).
As can be easily appreciated, however, neurotropic snake venom, such as derived from the cobra and krait snakes, is extremely toxic and must be carefully detoxified before administrating to humans. On the other hand, the detoxification procedure can also be carried to a point where the modified snake venom is not only detoxified, but to where its neurotropic character is also destroyed. Thus, if the modified neurotoxin is carried through the detoxification procedure to that extent, it will not function in the manner intended by the Sanders patent.
The preferred detoxification procedure is that of mild oxygenation of a solution of the snake venom with the procedure being carried to the end point of toxicity but before deterioration of the neurotropic character commences. The usual method of following the detoxification process is by bioassay with laboratory animals. This test will determine lack of toxicity, but it does not give any indication of the potency of the detoxified snake venom.
The Sanders patent discloses a method of determining potency of a detoxified venom by an adaptation of the Semliki Forest virus test. In that test, a sheet of chick embryo fibroblastic tissue cells on glass is covered with a gelled nutrient preparation, such as Hank's solution with lactalbumin. As a control, Semliki Forest virus is inoculated on the chick cells and the number of resulting plaques show the titer of the virus. In the test, the chick cells are washed with a detoxified venom prior to inoculation with the control virus and if the detoxified venom has acceptable potency the cells will show few or no plaques of viral growth. If, however, a substantial number of plaques are observed, the detoxification procedure practiced with the venom has been too severe or carried beyond the end point and unacceptable denaturization of the venom has occurred, i.e., the potency of the detoxified venom has been unacceptably reduced. The Sanders patent requires that the cells washed in the detoxified venom always show at least a statistically significant inhibition of plaque formation by the virus, e.g., 30%, especially 50% inhibition of the plaques and preferably at least 70% to 75% inhibition of the plaques.
While that Semliki Forest virus test does produce the results stated in the Sanders patent, the accuracy of the potency test is not as great as desired. Additionally, that test gives no indication of toxicity/atoxicity and the bioassay by animal test must be performed for that purpose. It would, therefore, be of substantial benefit in the art if the Semliki Forest virus test could be improved to give greater accuracy of the potency of the modified neurotoxin and at the same time at least give some indication of toxicity/atoxicity. This would materially aid in finding that delicate end point during the detoxification procedure.